Polysaccharides are high molecular weight (104-107) polymeric biomaterials, formed through the polymerization of monosaccharide repeating units. They possess a great structural diversity as a result of the diversity of the repeating units, type of glycosidic linkages involved and the degree of branching. Many polysaccharides possess non-sugar components, such as organic acyl groups (e.g. acetate, succinate, piruvate) and inorganic groups (e.g. phosphate, sulfate) (Sutherland, 2001).
On the other hand, polysaccharides often form tertiary structures through intra or intermolecular non-covalent linkages, which confer greater rigidity to the macromolecule and play an important role in determining the polymer's properties both in the solid state and in solution (Kumar et al, 2007).
Due to their physical and chemical properties, namely, their water retention ability, rheology and/or film-forming capacity, polysaccharides are used in a wide variety of foods and industrial applications, including textiles, paints, pharmaceuticals and cosmetics, as emulsifying, stabilizing or thickening agents (Moreno et al, 1998). Being materials obtained from living organisms, polysaccharides are usually non-toxic and biodegradable, which makes them biomaterials adequate for sustainable development.
The main applications of commercial polysaccharides, both natural (e.g. alginate, carrageenan, Guar gum, pectins, xanthan, gellan) and semi-synthetic derivatives (e.g. methylcellulose, carboxymethylcellulose, hydroxypropylguar) are based on their ability to modify the physical properties of aqueous systems (hydrocolloids—compounds able to modify the physical properties of aqueous systems), being used mainly in the food industry, followed by the oil and pharmaceuticals industries. Some of these polysaccharides (e.g. alginate, pectins, pullullan, starch derivatives, cellulose derivatives) additionally possess the capacity to form biodegradable films, being used in the manufacturing of packages, vessels and sheets, as well as in several agro-food, pharmaceuticals and industrial applications.
Currently, the majority of the polysaccharides used in industry are obtained from plants (e.g. Guar gum, Arabic gum), algae (e.g. alginate, carrageenan) or crustacean (e.g. chitin), with microbial polysaccharides (e.g. xanthan, gellan, bacterial alginate) representing only a small fraction of the biopolymer's market (Canilha et al, 2005). Nonetheless, in the last years, there has been a growing interest in identifying and isolating new microbial polysaccharides that may compete with the traditional ones, due to their enhanced physical-chemical properties, namely, higher emulsifying and flocculating activities, higher resistance to organic solvents, biological activity (e.g. anticancer or immunoenhancing effects) and better rheological properties (e.g. higher viscosity for lower polymer concentrations, higher stability over wider pH, temperatures and ionic strength ranges) (Kumar et al, 2007; Sutherland, 2001).
Microbial production of polysaccharides has advantages over their extraction from plants, algae or animals, since microorganisms usually exhibit higher growth rates and they are more amenable to manipulation of the cultivation conditions (Moreno et al, 1998). Plants, algae and animals have life cycles of one or more years, being the production cycle usually seasonal. On the other hand, the growth rates of microorganisms is of the order of hours or a few days, while plants, algae and animals have growth rates of the order of months or years.
The main factor limiting the commercial production of microbial polysaccharides is the high substrate cost, mainly sugars, especially glucose, starch and sucrose. In those bioprocesses, substrate cost accounts to 20-40% of the total production costs (Kumar and Mody, 2009).
Hence, the search for less expensive substrates with comparable productivity is essential for the reduction of the production costs. Glycerol, a byproduct of several industrial processes, mainly the biodiesel industry, is a good candidate. Due to the huge growth of the biodiesel industry in the last years, it is being produced in quantities far beyond its current consumption in the traditional glycerol applications. For the biodiesel industry or for any other industry that has glycerol as a byproduct, it represents a burden because of its low commercial value and the fact that its elimination is a cost associated process. Therefore, there is an urgent need for the development of interesting application for this industrial byproduct, making use of the fact that glycerol is a non-toxic and biodegradable compound (Celik et al, 2008).
In addition to their ability to modify the physical properties of aqueous systems, fucose-containing polysaccharides have increased potential for industrial applications due to the fact that fucose is one of the rare sugars, difficult to obtain. On the other hand, the presence of fucose reduces the possibility of allergic reactions, which potentiates the use of these biopolymers in application such as, for example pharmaceuticals and cosmetics.
Fucose may be synthesized through its chemical conversion from other more common monosaccharides, such as galactose or glucose. Nevertheless, most chemical processes are complex, involving several intermediates, and have low yield. An alternative to the chemical synthesis of fucose is the chemical or enzymatic hydrolysis of fucose-containing polysaccharides. These polymers may be found in plants, algae and microorganisms.
In plants, fucose (L-fucose and methylated fucose) occurs, for example, in the cells walls of potato and kiwi fruit, in soybean seeds, in the mucilage of young leaves of Plantago lanceolata, in the roots of Lepidium sativum and Glycyrrhiza uralensis, in the exudates of Astragalus microcephalus, A. gummifer and A. kurdicus, and in the leafs of Lupinus albus (Vanhooren e Vandamme, 1999).
In seaweeds, fucose is found in fucoidan that is a homopolysaccharide composed of sulfated L-fucose. Fucose may be extracted from seaweeds such as, for example, Pelvetia canaliculata, Fucus vesiculosus and Ascophyllum nodosum. In those species, L-fucose content varies between 9.0 and 11.2%. The yield of the global extraction of L-fucose from seaweeds is rather low (around 7.6%) (Vanhooren e Vandamme, 1999.
Several microorganisms, namely, bacteria, fungi and microalgae, synthesize extracellular polysaccharides (EPS) that contain L-fucose. These polymers include both homo and heteropolysaccharides, being the later are more common, containing variable amounts of fucose, as well as other sugar residues (e.g. glucose, galactose, mannose, rhamnose and/or arabinose). L-fucose containing EPS are produced by bacteria belonging to several genera, including Aerobacter, Azotobacter, Klebsiella, Erwinia, Enterobacter, Pseudomonas, Clavibacter, Bacillus and Salmonella, among others. In fungi, fucose may be found in EPS produced by species belonging to the genera Candida, Mucor, Polyporus, Rhodotorula e Sporobolomyces, among others.
In the last decades, the production of L-fucose containing polysaccharides has been reported for several bacterial genera, mainly from the genera Klebsiella, Enterobacter, Pseudomonas and Clavibacter. 
Several Klebsiella pneumoniae strains synthesize several different EPS containing L-fucose, D-galactose and galacturonic acid, that differ among them by the degree of acetylation of the polymeric chain. The EPS produced by K. pneumoniae 1-1507 (U.S. Pat. No. 5,876,982) has found application in the cosmetics industry due to its psychosensorial qualities, hydrating and self-emulsifying properties (Guetta et al, 2003a). Other EPS possessing a very similar composition, have been described, namely, the EPS produced by Klebsiella K-63 (Joseleau and Marais, 1979) and by K. pneumoniae ATCC 31646 (U.S. Pat. No. 4,298,691). The polymer of this disclosed subject matter differs from those EPS by the fact that, in addition to fucose and galactose, it also contains glucose. On the other hand, the polymer of this disclosed subject matter has in its composition significant amounts of acyl groups substituents (up to about 25% of the EPS dry weight) that are not referred as components of Klebsiella EPS.
K. pneumoniae ATCC 12657 (formerly known as Aerobacter aerogenes strain A3) produces an EPS composed of fucose, glucose, galactose and glucuronic acid, in approximately equimolar amounts (Vanhooren e Vandamme, 1999). Fucose represents 18.9% of the purified EPS weight (Guetta et al, 2003b). This polysaccharide differs from the polymer of this disclosed subject matter by the high glucuronic acid content, and the presence of acyl groups that are not described for K. pneumoniae ATCC 12657 EPS. Moreover, the process described in the presently disclosed subject matter does not use species of the Klebsiella genus for the microbial cultivation.
Enterobacter strains have also been reported to produce EPS containing fucose, galactose, glucose and glucuronic acid. Examples include: Enterobacter sp. CNCM 1-2744 that produces an EPS in which the monomers are present in a ratio of 2:2:1:1 (FR2840920); Enterobacter sp. SSYL (KCTC 0687BP) that produces an EPS with a molecular weight between 105 and 106, in which fucose represents 8-10% of the sugar content, being glucuronic acid the main component (40-70%) (US2002115158); and E. sakazakii, strains ATCC 53017, ATCC 29004 and ATCC 12868 that produces an EPS with a molecular weight of 2×106, in which fucose represent 13-22% and mannose content is up to 8%, respectively (U.S. Pat. No. 4,806,636). These polysaccharides differ from the polymer of this disclosed subject matter by the different content of their sugar monomers and acyl groups. Additionally, the polymer of this disclosed subject matter tends to have a typically higher molecular weight in the order of 106-107.
Several Clavibacter michiganensis strains have been described that produce EPS containing L-fucose. Those EPS contain other neutral sugars, such as galactose, glucose and/or mannose, and acyl groups substituents, such as pyruvate, succinate and/or acetate. C. michiganensis subsp michiganensis Cm 542 (NCPPB 1064) produces a high molecular weight EPS (106-107) composed by fucose, galactose and glucose (2:1:1), and pyruvate, succinate and acetate (1:0.5:1.5) (van den Bulk et al, 1991). The polymer of this disclosed subject matter, though possessing a similar composition, differs from C. michiganensis EPS by the relative proportion of the acyl groups. The higher succinate content of the polymer of this disclosed subject matter confers it a higher anionic character.
In the document WO2008/127134 a process is described for the production of a galactose-rich polysaccharide by the bacterium Pseudomonas oleovorans using glycerol rich substrates. Nevertheless, the EPS obtained in that process contains only residual amounts of fucose (0-4%).
In view of this, the presently disclosed subject matter describes a high molecular weight fucose-containing biopolymer, with a polyelectrolyte character, produced by microbial cultivation, preferably using Enterobacter A47 (DSM 23139) and a process thereof. Said process allows obtaining the polymer of the disclosed subject matter using low-cost substrates and in an easy way. The polymer of the disclosed subject matter may be used in several industries, such as agro-food industry, waste water treatment and pharmaceutical industry due to its rehology, film-forming capacity, polyelectrolyte character, and emulsifying flocculating abilities.
General Description of the Disclosed Subject Matter
The presently disclosed subject matter concerns the production of a biopolymer whose main component is a high molecular weight polysaccharide (106-107), in which fucose represents at least 10% of its composition, and possessing acyl groups substituents, including pyruvate, succinate and acetate. The biopolymer is obtained by microbial cultivation, preferably by the bacterium Enterobacter A47 (DSM 23139) using glycerol or glycerol containing substrates as the preferential carbon sources.
Accordingly, the presently disclosed subject matter provides a process for preparing a polymer comprising the steps of cultivating a microbial culture comprising the bacterial strain Enterobacter A47 (DSM 23139) and supplying said culture with a carbon source comprising glycerol.
1. Characterization of the Microbial Culture
The fucose-containing polymer of the presently disclosed subject matter is produced by bacteria of the genera Pseudomonas, Klebsiella, Methylobacterium, Erwinia, Alcaligenes or Enterobacter, preferably by the bacterium Enterobacter A47 deposited Nov. 20, 2009 in DSMZ (Deutsche Sammlung von Mikroorganismen and Zellkulturen GmbH), Inhoffenstr. 7B, D-38124 Braunschweig, Germany, under the Budapest Treaty, with accession number DSM 23139. In addition, the microorganism used in the presently disclosed subject matter is characterized by other aspects, namely, the biochemical profile, genetic sequencing and phylogenetic dendrogram presented in Tables 1 and 2, and FIG. 1, respectively.
The microorganism used in this disclosed subject matter may be a wildtype strain, a variant or a mutant, as long as it possesses the ability to synthesize the fucose-containing polymer. It is possible to use a pure culture or a mixed culture of several microorganisms, among which, at least one is capable of producing the fucose-containing polymer, preferably the bacterium Enterobacter A47 (DSM 23139).
2. Characterization of the Process for the Production of the Polymer
The polymer of the presently disclosed subject matter is produced in a bioreactor in a stirred and aerated aqueous medium. The cultivation medium contains a carbon source, a nitrogen source and inorganic salts. The preferential carbon source is glycerol or glycerol containing substrates. Nevertheless, the process for the production of the polymer of the disclosed subject matter foresees the use of other carbon sources, either in alternative to glycerol or in mixture with glycerol, such as for example, sugars, alcohols, organic acids or alkanes, as well as food and industrial wastes or byproducts, such as for example glycerol byproduct from the biodiesel industry, sugar molasses, whey or olive oil production wastes.
The process for the production of the fucose-containing polymer consists on the cultivation of the microorganism in a nutrient aerated aqueous medium. The temperature is controlled between 15 and 45° C., preferably between 26 and 37° C. The pH is controlled between 5.0 and 9.0, preferably between 6.5 and 7.0.
At the beginning of the cultivation, the dissolved oxygen concentration in the cultivation medium is settled above 70%. Afterwards, the dissolved oxygen concentration gradually decreases, concomitant with cell growth, being controlled below 30%, or preferably below 20%, or most preferably below 10% or even in anaerobic conditions. The fucose-containing polymer is produced under conditions of nitrogen limitation, such as in an amount less than 0.3 g/L, or less than 0.2 g/L, or less than 0.1 g/L or even without nitrogen source and carbon availability, simultaneously with low dissolved oxygen concentration, as described above. Carbon availability is guaranteed by supplying the culture with cultivation medium containing a high glycerol concentration (>100 g/L). The flow rate of addition of such a medium during this fed-batch phase must be adjusted to match the culture's carbon consumption.
The culture broth obtained at the end of the cultivation in the bioreactor may be used directly, without any treatment, or after being dried. Alternatively, the fucose-containing polymer may be precipitated from the broth by the addition of a precipitating agent (e.g. ethanol, acetone), yielding a native polymer.
The extraction process of the polymer of the disclosed subject matter consists on cell removal (e.g. by centrifugation of the broth), followed by the precipitation of the polymer by addition of a precipitating agent. The purification of the polymer involves the use of one or several additional processes (e.g. dialysis).
Depending of the cultivation conditions in the bioreactor, the cultivation time and the procedures used to extract/purify the polymer, the process yields 50 g/L of native polymer or 20 g/L of purified polymer.
3. Characterization of the Polymer
Typically, the polymer of the disclosed subject matter has a fucose content that represents at least 10% of its sugar composition. The fucose-containing polymer has in its composition other neutral sugars, namely, glucose and galactose, and it may also contain in trace amounts (<5%) other sugars, such as for example mannose, rhamnose, arabinose, fructose, glucuronic acid and/or glucosamine.
4. Applications of the Polymer
The polymer of the disclosed subject matter presents flocculating and emulsifying activities, it forms highly viscous aqueous solutions with pseudoplastic fluid behavior, and produces biodegradable films when mixed with other polymers. Hence, it may replace other polysaccharides, such as for example xanthan, alginate, carrageenan, Guar gum and Arabic gum, in their numerous applications, namely, in the agro-food industry, in pharmaceuticals and cosmetics. In addition, the presence of fucose in the polymer of the disclosed subject matter further potentiates its use in medical and cosmetic applications. Moreover, the presence of pyruvate and succinate residues confers an anionic character to the polymer. As a consequence, it is able to immobilize toxic metals.